Acetyl-coenzyme a carboxylase 2 as a target in the regulation of fat burning, fat accumulation, energy homeostasis and insulin action

ABSTRACT

The present invention highlights the role of acetyl-CoA carboxylase through its product malonyl-CoA in regulating fatty acid oxidation and synthesis, glucose metabolism and energy homeostasis. It discloses transgenic mice with inactivating mutations in the endogenous gene for the acetyl-CoA carboxylase 2 isoform of acetyl-CoA carboxylase. Inactivation of acetyl-CoA carboxylase 2 results in mice exhibiting a phenotype of reduced malonyl-CoA levels in skeletal muscle and heart, unrestricted fat oxidation, and reduced fat accumulation in the liver and fat storage cells. As a result, the mice consume more food but accumulate less fat and remain leaner than wild-type mice fed the same diet. These results demonstrate that inhibition of ACC2 acetyl-CoA carboxylase could be used to regulate fat oxidation and accumulation for purposes of weight control. The instant invention provides a useful animal model to regulate malonyl-CoA production by ACC2 in the regulation of fatty acid oxidation by muscle, heart, liver and other tissues. They also identify potential inhibitors for studying the mechanisms of fat metabolism and weight control.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This non-provisional patent application is a continuation in partof, claims benefit of, U.S. Ser. No. 09/749,109, filed Dec. 26, 2000.

FEDERAL FUNDING LEGEND

[0002] This invention was produced in part using funds from the Federalgovernment under N.I.H. G.M. 19091. Accordingly, the Federal governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the field of fatmetabolism and weight control. More specifically, the present inventionrelates to the role of the ACC2 isoform of acetyl-CoA carboxylase inregulating fatty acid accumulation and oxidation.

[0005] 2. Description of the Related Art

[0006] Acetyl-CoA carboxylase (ACC), a biotin-containing enzyme,catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, anintermediate metabolite that plays a pivotal role in the regulation offatty acid metabolism. It has been found that malonyl-CoA is a negativeregulator of carnitine palmitoyltransferase I (CPTI, a component of thefatty-acid shuttle system that is involved in the mitochondrialoxidation of long-chain fatty acids. This finding provides an importantlink between two opposed pathways-fatty-acid synthesis and fatty-acidoxidation. Thus, it is possible to interrelate fatty acid metabolismwith carbohydrate metabolism through the shared intermediate acetyl-CoA,the product of pyruvate dehydrogenase. Consequently, the roles ofmalonyl-CoA in energy metabolism in lipogenic (liver and adipose) andnon-lipogenic (heart and muscle) tissues has become the focus of manystudies.

[0007] In prokaryotes, acetyl-CoA carboxylase is composed of threedistinct proteins-the biotin carboxyl carrier protein, the biotincarboxylase, and the transcarboxylase. In eukaryotes, however, theseactivities are contained within a single multifunctional protein that isencoded by a single gene.

[0008] In animals, including humans, there are two isoforms ofacetyl-CoA carboxylase expressed in most cells, ACC1 (M_(r)˜265,000) andACC2 (M_(r)˜280,000), which are encoded by two separate genes anddisplay distinct tissue distribution. Both ACC1 and ACC2 producemalonyl-CoA, which is the donor of the “C₂-units” for fatty acidsynthesis and the regulator of the carnitine palmitoyl-CoA shuttlesystem that is involved in the mitochondrial oxidation of long-chainfatty acids. Hence, acetyl-CoA carboxylase links fatty acid synthesisand fatty acid oxidation and relates them with glucose utilization andenergy production, because acetyl-CoA, the substrate of thecarboxylases, is the product of pyruvate dehydrogenase. Thisobservation, together with the finding that ACC1 is highly expressed inlipogenic tissues such as liver and adipose and that ACC2 ispredominantly expressed in heart and skeletal muscle, opened up a newvista in comparative studies of energy metabolism in lipogenic and fattyacid-oxidizing tissues.

[0009] Diet, especially a fat-free one, induces the synthesis of ACC'sand increases their activities. Starvation or diabetes mellitusrepresses the expression of the Acc genes and decreases the activitiesof the enzymes. Earlier studies addressed the overall activities of thecarboxylases with specific differentiation between ACC1 and ACC2.Studies on animal carboxylases showed that these enzymes are underlong-term control at the transcriptional and translational levels andshort-term regulation by phosphorylation/dephosphorylation of targetedSer residues and by allosteric modifications induced by citrate orpalmitoyl CoA.

[0010] Several kinases have been found to phosphorylate bothcarboxylases and to reduce their activities. In response to dietaryglucose, insulin activates the carboxylases through theirdephosphorylation. Starvation and/or stress lead to increased glycogenand epinephrine levels that inactivate the carboxylases throughphosphorylation. Experiments with rats undergoing exercises showed thattheir malonyl-CoA and ACC activities in skeletal muscle decrease as afunction of exercise intensity thereby favoring fatty acid oxidation.These changes are associated with an increase in AMP-kinase activity.The AMP-activated protein kinase (AMPK) is activated by a high level ofAMP concurrent with a low level of ATP through mechanism involvingallosteric regulation and phosphorylation by protein kinase (AMP kinase)in a cascade that is activated by exercise and cellular stressors thatdeplete ATP. Through these mechanisms, when metabolic fuel is low andATP is needed, both ACC activities are turned off by phosphorylation,resulting in low malonyl-CoA levels that lead to increase synthesis ofATP through increased fatty acid oxidation and decreased consumption ofATP for fatty acid synthesis.

[0011] Recently, it was reported that the cDNA-derived amino acidsequences of human ACC1 and ACC2 share 80% identity and that the mostsignificant difference between them is in the N-terminal sequence ofACC2. The first 218 amino acids in the N-terminus of ACC2 represent aunique peptide that includes, in part, 114 of the extra 137 amino acidresidues found in this isoform. Polyclonal antibodies raised against theunique ACC2 N-terminal peptide reacted specifically with ACC2 proteinsderived from human, rat, and mouse tissues. These findings made itpossible to establish the subcellular localization of ACC1 and ACC2 andto later demonstrate that ACC2 is associated with the mitochondria andthat the hydrophobic N-terminus of the ACC2 protein plays an importantrole in directing ACC2 to the mitochondria. ACC1, on the other hand, islocalized to the cytosol.

[0012] Although these findings and the distinct tissue distribution ofACC1 and ACC2 suggest that ACC2 is involved in the regulation of fattyacid oxidation and that ACC1 is involved in fatty acid synthesisprimarily in lipogenic tissues, they do not provide direct evidence thatthe products of the genes ACC1 and ACC2 have distinct roles.

[0013] These distinctions between the two ACC isoforms could not havebeen predicted prior to the generation of the Acc2 knockout mousedescribed herein. Moreover, malonyl-CoA, the product of the ACC1 andACC2, seems to be present in the liver and possibly in other tissues intwo separate pools that do not mix and play distinct roles in thephysiology and metabolism of the tissues. Malonyl-CoA, the product ofACC1, is involved in fatty acid synthesis as the donor of “C2-carbons.”On the other hand, malonyl-CoA, the product of ACC2, is involved in theregulation of the carnitine palomitoyl CoA shuttle system, hence in theoxidation of fatty acids. This functional distinction between the rolesof the products of ACC1 and ACC2 based on the results obtained with theAcc2 mice was not expected nor could it have been predicted prior tothis study.

[0014] Moreover, the current study demonstrates that ACC2, through itsproduct malonyl-CoA, is potentially an important target for theregulation of obesity. Inhibition of ACC2 would reduce the production ofmalonyl-CoA, leading to continual fatty acid oxidation and energyproduction. This continual oxidation of fatty acid would be achieved atthe expense of freshly synthesized fatty acids an d triglycerides and ofbody fat accumulated in the adipose and other fatty tissues leading toreduced body fat.

[0015] The prior art is deficient in an understanding of the separateroles ACC1 and ACC2 have in the fatty acid metabolic pathways. The priorart is also deficient in assigning the differential roles of themalonyl-CoA generated by ACC1 versus that generated by ACC2 inregulating fatty acid metabolism. Also, the prior art is deficient intransgenic knockout mice generated to lack ACC2 and methods of usingthese transgenic mice. The present invention fulfills this long-standingneed and desire in the art.

SUMMARY OF THE INVENTION

[0016] Malonyl-CoA (Ma—CoA), generated by acetyl-CoA carboxylases ACC1and ACC2, is the key metabolite in the regulation of fatty acid (FA)metabolism. Acc1^(−/−) mutant mice were embryonically lethal, possiblydue to a lack of “C₂-units” for the synthesis of fatty acid needed forbiomembrane synthesis. Acc2^(−/−) mutant mice bred normally and hadnormal life spans. Acc2^(−/−) mice fed normal diets did not accumulatefat in their livers as did the wild-type mice and overnight fastingresulted in a 5-fold increase in ketone bodies production, indicatinghigher fatty acid oxidation. ACC1 and fatty acid synthase activities andmalonyl-CoA contents of the livers of the Acc2^(−/−) and Acc2^(+/+) micewere the same, indicating that fatty acid synthesis is unperturbed, yetthe malonyl-CoA was not available for the inhibition of themitochondrial fatty acid shuttle system, hence fatty acid oxidation wasrelatively high. This result was not predicted earlier to this finding,and it is very important in distinguishing the roles of the malonyl-CoAgenerated by ACC1 versus that generated by ACC2 in regulating fatty acidmetabolism.

[0017] Absence of ACC2 resulted in 30- and 10-fold lower malonyl-CoAcontents of muscles and heart, respectively. Fatty acid oxidation in theAcc2^(−/−) soleus muscles was 30% higher than that of ACC2^(+/+) mice.Addition of insulin did not affect fatty acid oxidation in theAcc2^(−/−) soleus muscle, but, as expected, it did reduce fatty acidoxidation by 50% in the wild-type soleus muscle compared to that of themutant. This is a very important observation since it demonstrates forthe first time the role of ACC2 in insulin action and regulation offatty acid oxidation in diabetes. Isoproterenol, an analog of glucagon,had little effect on fatty acid oxidation in the muscle of theAcc2^(−/−) mice but caused a 50% increase in fatty acid oxidation in thesoleus muscle. Again, this result highlights the important role of ACC2in regulating fatty acid oxidation and its potential as a target for theregulation of obesity. The higher fatty acid oxidation in the mutantmice resulted in a 50% reduction of fat storage in the adipose tissuecompared to that of the wild-type mice. These results are valuable to anunderstanding and control of fatty acid metabolism and energyhomeostasis in normal, diabetic, and obese animals, including humans.

[0018] In one embodiment of the instant invention, a method of promotingweight loss and/or fat oxidation in an individual is provided. Thismethod may comprise the administration of an inhibitor of acetyl-CoAcarboxylase 2 (ACC2) to the individual. The same method may be used forweight loss as well.

[0019] In yet another embodiment of the instant invention, a method isprovided for promoting fatty acid oxidation to treat conditions such asobesity and diabetes comprising the administration of an inhibitor ofacetyl-CoA carboxylase 2 (ACC2) to an individual having such conditions.

[0020] In another embodiment of the instant invention, a method ofdecreasing blood sugar by administering an inhibitor of acetyl-CoAcarboxylase 2 (ACC2) to an individual is provided. This method may beused to treat an individual with diabetes.

[0021] In another embodiment of the present invention, there is provideda transgenic mouse having a mutation in an endogenous gene for the ACC2isoform of acetyl-CoA carboxylase that inactivates the protein. The ACC2gene may be mutated by deleting one or more exons of the gene, which maybe replaced by heterologous DNA sequences such as an HPRT expressioncassette. In a preferred embodiment, an exon encoding a biotin-bindingmotif of ACC2 is replaced with an HPRT expression cassette. Unexpectedlyto those in the field, these mice exhibit a phenotype consisting of areduction in malonyl-CoA levels in skeletal muscle and heart,unrestricted fat oxidation, and reduced fat accumulation in the liverand fat storage cells. The transgenic mice consume more food thanwild-type mice but remain lean.

[0022] In yet another embodiment of the present invention, there isprovided a method of screening for an inhibitor of ACC2 isoform activityconsisting of the step of administering potential inhibitors towild-type mice and screening for mice that exhibit the same phenotype ofthe Acc2^(−/−) transgenic mice.

[0023] In yet another embodiment of the present invention, there isprovided an ACC2 inhibitor identified by the above method. Thisinhibitor may be incorporated into a pharmaceutical composition to beadministered to individuals for purposes of augmenting fatty acidoxidation and inhibiting fat accumulation to promote weight loss ormaintenance.

[0024] The present invention has further potential for the treatment ofdiabetic animals, including humans, in that it may helpinsulin-administered type I and type II diabetics from gaining weight.Furthermore, increased fatty acid oxidation would affect carbohydratemetabolism by increasing glycolysis, and reducing gluconeogenesis andglycogen synthesis and accumulation of fatty acid oxidation independentof insulin. Thus it helps diabetics to burn fat and lose weight.

[0025] In a further embodiment of the instant invention, a method isdescribed for obtaining a purified preparation of ACC1 protein totallyfree of the ACC2 isoform by purifying ACC1 from the Acc2^(−/−)transgenic mice.

[0026] In another embodiment of the instant invention, a method isprovided for obtaining improved antibodies against ACC2 by generatingthe antibodies in the Acc2^(−/−) transgenic mice.

[0027] In yet another embodiment of the instant invention, cell linesderived from the Acc2^(−/−) transgenic mice are provided. Cell linesderived from muscle, heart, adipose cells, and liver cells are expectedto be especially useful in bioassays and drug targeting studies. Braincell lines including those of the hypothalamus would be useful instudying the neuropeptides involved in regulating feeding behavior andappetite and fat and carbohydrate metabolism.

[0028] In yet another embodiment of the present invention, a method ofscreening for agonists and antagonists of ACC2 is provided. This methodcomprises the steps of administering candidate compounds to Acc2^(−/−)cell lines and to cell lines derived from wild-type mice followed byexperiments to detect alterations in cellular activity. A compound thatspecifically acts on ACC2 will alter cellular activity, fat andcarbohydrate metabolism in wild-type cells but have no effect onAcc2^(−/−) cells. Cellular activities that may be monitored include mRNAexpression, protein expression, protein secretion, and catalyticallyactive proteins (enzymes) involved in fatty acid and lipid andcarbohydrate metabolism.

[0029] The absence of Ser 1201 in ACC2 represents an importantdifference between ACC1 and ACC2 regulation and can b e advantageous indesigning and/or generating differential inhibitor(s) [drug(s)] for ACC1and ACC2. Other and further aspects, features, and advantages of thepresent invention, including the unique hydrophobic amino-terminal ofACC2, will be advantageous in designing and/or generating differentialinhibitor(s) [drug(s)] for ACC1 and ACC2. Also, the differentialreactions of ACC2 to anti-ACC1 antibodies would be important indesigning and generating differential inhibitors for ACC1 and ACC2.Moreover, further aspects will be apparent from the followingdescription of the embodiments of the invention given for the purpose ofdisclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others that willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustrateembodiments of the invention and therefore are not to be consideredlimiting in their scope.

[0031]FIG. 1A shows the strategy used in the targeted mutation of theAcc2 locus. Of the two exons (dark boxes) that were identified in themouse genomic clone, the exon that contained the biotin-binding motif(Met-Lys-Met) was replaced with a hypoxanthinephosphorylribosyltransferase (HPRT) expression cassette to generate thetargeting construct. The 3′ and 5′ probes used to identify the targetedevents by Southern blot analysis are indicated.

[0032]FIG. 1B shows a Southern blot analysis of the genomic DNAsextracted from mouse tails. DNA's that were digested with BglI wereprobed with the 5′ probe; the DNAs digested with Bam H1 and Kpn 1 wereprobed with the 3′ probe. DNAs from the wild-type (+/+), heterozygous(+/−), and Acc2-null (−/−) mice gave the expected fragment sizes.

[0033]FIG. 1C shows a Northern blot of total RNA prepared from theskeletal muscles of wild-type (+/+), heterozygous (+/−), and Acc2-null(−/−) mice was probed with the ³²P-labeled 362-bp cDNA fragment, whichwas used to screen the genomic library. The probe detected a 10-kbp RNAband in the Acc2^(+/−) and Acc2^(+/+) RNAs but not in the Acc2^(−/−)RNA. Hybridization of the same filter (after stripping) with a mouseβ-actin cDNA probe confirmed that equal amounts of RNA were loaded inthe gel.

[0034]FIG. 1D shows a confirmation of the absence of ACC2 protein in theAcc2-null mice. Extracts (50 μg each) from the livers, skeletal muscles,and hearts of the mice were separated by SDS-PAGE (6%). The proteinswere transferred onto a nitrocellulose filter and probed withavidin-peroxidase to detect biotin-containing proteins. The locations ofthe two carboxylases—the 280-kDa ACC2 and the 265-kDa ACC1 —areindicated.

[0035]FIG. 2 shows the relative amounts of malonyl-CoA in the tissues ofwild-type (filled symbol) and Acc2^(−/−) mutant (open symbol) mice.Malonyl-CoA in the acid-soluble extracts of the indicated mouse tissueswas measured by the incorporation of [³H]acetyl-CoA into palmitate inthe presence of reduced nicotinamide adenine dinucleotide phosphate(NADPH) and highly purified chicken fatty acid synthase (4,29). The[³H]palmitic acid synthesized was extracted with petroleum ether and theradioactivity was measured. The mice were either fed normal chow or werefasted for 48 hours before they were sacrificed. The data are mean±SDfrom three animals.

[0036] FIGS. 3A-3E show histological analyses of livers of 32-week-oldmale mice fed a standard diet. FIG. 3A shows livers of wild-type (left)and Acc2^(−/−) mutant mice right after 24 hours of starvation. Frozensections of wild-type and mutant livers were stained with Oil Red-O todetect lipid droplets and counter-stained with Mayer's hematoxylin. Theliver sections of wild-type mice (FIG. 3B) show an abundance ofred-stained lipid droplets compared to the dramatic decrease inred-stained droplets in the Acc2^(−/−) mutant liver (FIG. 3C). Frozensections were made from the same livers and stained for glycogen by theperiodic acid-Schiff method and counter-stained with hematoxylin. Thewild-type livers (FIG. 3D) contain glycogen (pink-stained) and unstainedlipid vacuoles, whereas the mutant livers (FIG. 3E) have little or noglycogen and few lipid vacuoles.

[0037]FIG. 4 shows a summary of an experiment in which mice weresacrificed by cervical dislocation, and the soleus muscles—two from eachhind limb—were resected from each mouse and were immersed in 1.5 ml ofKrebs-Henseleit buffer (pH 7.4) containing 4% fatty acid-free bovineserum albumin, 10 mM glucose, and 0.3 mM [9,10(n)-³H]palmitate (3mCi/vial) [Ibrahimi, 1999 #423]. Where indicated, insulin (10 nM) orisoproterenol (3 mM) was added, and the vials were incubated at 37° C.under a humidified O₂/CO₂ (95/5%) atmosphere for 30 min. At the end ofthe incubation period, the [³H]₂O was separated from the labeledsubstrate and counted.

[0038] FIGS. 5A-5E show food intake, growth (body weight) and adiposetissue in Acc2^(−/−) and wild-type mice. Two groups of female mice(numbered 1 and 2; 3 and 6 weeks old, respectively) and one group of5-week-old males—each group consisting of five Acc2^(−/−) mutants (M,filled circles) and five wild type (W, open symbols)—were fed a standarddiet for 27 weeks. In FIG. 5A, food intake was measured every week andexpressed as cumulative food intake per mouse over the 27-week period.The weight of each mouse within each group was measured weekly and thedata are presented as means±SD in FIG. 5B. FIG. 5C shows dorsal views ofmale littermates, aged 32 weeks, fed with standard diet. The amount ofwhite fat observed under the skin of the Acc2^(−/−) mouse (33.6 gweight) was much less than that of the wild-type mouse (34.2 g weight).FIG. 5D shows an abdominal view of the fat pads under the skin ofAcc2^(−/−) and wild-type mice (+/+). FIG. 5E shows epididymal fat padsisolated from the mutant (0.75 g) and wild-type (1.4 g) mice. Bar, 1 cm.

[0039]FIGS. 6A and 6B show the targeted mutation of the Acc1 locus. FIG.6A shows the strategy used to create the targeted mutation. The exon(dark box) that contains the biotin-binding motif (Met-Lys-Met) wasreplaced with an HPRT expression cassette. The 3′ and 5′ probes used forSouthern blot analysis are indicated. FIG. 6B shows a typical patternobserved in genotyping by Southern blot analyses of genomic DNAextracted from mouse tails. The DNAs were digested with ShpI induplicate. The blots were probed with the 5′ and 3′ probes indicated inFIG. 6A. The presence of only wild-type (+/+) and heterozygous (+/−)genotypes indicated that no homozygous (−/−) mice were born.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The instant invention is directed to a method of promoting weightloss in an individual by administering an inhibitor of acetyl-CoAcarboxylase 2 (ACC2) to said individual. The same method may be used forfat reduction as well.

[0041] The instant invention provides a method of promoting fatty acidoxidation to treat conditions such as obesity and diabetes byadministering an inhibitor of acetyl-CoA carboxylase 2 (ACC2) to anindividual having such conditions.

[0042] The present invention provides a method of decreasing anindividual's blood sugar levels by administering an inhibitor ofacetyl-CoA carboxylase 2 (ACC2) to the individual. This method may beused to treat an individual with diabetes.

[0043] The present invention also provides a transgenic mouse having amutation in an endogenous ACC2 gene for the ACC2 isoform of acetyl-CoAcarboxylase, which results in the lack of expression of a functionalACC2 isoform. This gene may be mutated by deleting one or more exons ofthe ACC2 gene, which may be replaced by heterologous DNA sequences suchas an HPRT expression cassette. Preferably, an exon encoding a biotinbinding motif of ACC2 is replaced with an HPRT expression cassette. Theresulting mice exhibit a phenotype consisting of a reduction inmalonyl-CoA levels produced by ACC2 in skeletal muscle, heart and allother tissues, unrestricted fat oxidation, and reduced fat accumulationin the liver and fat storage cells. The transgenic mice consume morefood than wild-type mice but accumulate less fat.

[0044] The present invention also demonstrates a method of screening foran inhibitor of ACC2 isoform activity consisting of administeringpotential inhibitors to wild-type mice and screening for mice whichexhibit the phenotype of the Acc2^(−/−) transgenic mice.

[0045] The present invention is also directed to an ACC2 inhibitoridentified by the above method. This inhibitor may be incorporated intoa pharmaceutical composition to be administered to individuals forpurposes of augmenting fatty acid oxidation and inhibiting fataccumulation to promote weight loss or maintenance.

[0046] The instant invention also provides a purification method forobtaining ACC1 protein that is free of the ACC2 isoform. This isaccomplished by purifying ACC1 from tissue obtained from the Acc2^(−/−)transgenic mice of the instant invention that lack the ACC2 isoform.

[0047] The instant invention also provides for the preparation ofimproved antibodies against ACC2 by generating the antibodies in theAcc2^(−/−) transgenic mice. Unlike wild-type mice, these mice are lessimmunologically tolerant of ACC2 since it is not present during thedevelopment of immunological self-tolerance. As a result, antibodiesobtained from immunization of the Acc2^(−/−) transgenic mice with ACC2are more directed to unique antigenic domains of ACC2 than similarantibodies generated in wild-type mice.

[0048] The instant invention is further directed to cell lines derivedfrom the Acc2^(−/−) transgenic mice. These cell lines are useful inbioassays of ACC1 and ACC2 and in drug targeting studies. Cell linesderived from the muscle, heart, adipose, and liver tissues areespecially useful in these studies.

[0049] The instant invention also includes a method of screening foragonists and antagonists of ACC2. Candidate compounds are administeredto both Acc2^(−/−) cell lines and wild-type cell lines. The cells arethen monitored for alterations in cellular function such as a mRNAexpression, protein expression, protein secretions, protein activities,and lipid metabolism. A compound that specifically acts on ACC2 willhave altering cellular activity in wild-type cells but will have noeffect on the Acc2^(−/−) cell line.

[0050] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1

[0051] Generation of Acc2^(−/−) Transgenic Mice

[0052] A mouse Acc2 genomic clone was isolated using an Acc2 cDNA probe.Based on the homology between the human and mouse ACC2 genes(Abu-Elheiga, L., Almarza-Ortega, D. B., Baldini, A., and Wakil, S. J.,J Biol Chem. 272, 10669-10677, 1997), two oligonucleotides from thebiotin-binding region based on the cDNA sequence of human ACC2 weredesigned: a forward primer (5′-CTGAATGATGGGGGGCTCCTGCTCT-3′; nucleotides2551-2575) (SEQ ID No. 1) and a reverse primer(5′-TTCAGCCGGGTGGACTTTAGCAAGG-3′; nucleotides 2890-2913) (SEQ ID No. 2).These primers were used to amplify cDNA from a Quick-Clone mouse heartCDNA pool (Clontech) template.

[0053] The cDNA fragment obtained was sequenced and used to screen a129/SvEv mouse genomic library to isolate a 16-kbp λ genomic clone. Bydigesting the 16-kbp λ genomic clone with different restriction enzymes,a restriction map was established and a gene targeting vectorconstructed that contained positive-negative selection markers andlacked the exon that contains the biotin-binding motif Met-Lys-Met (FIG.1A). This vector was used to generate murine 129SvEv ES cells with onemutant copy of ACC2 gene (the mutant allele was termed Acc2^(tm1 LAE)).

[0054] Two independent ES-cell clones were injected into mouseblastocysts, which were then implanted into the uterine horns ofpseudopregnant females. Among the pups produced, eight high-levelchimeras were identified and crossbred with C57BL/6J females. Eachfemale gave birth to several agouti pups, indicating germ-linetransmission of the ES-cell genome. Southern blot analysis of genomicDNA confirmed the presence of both the endogenous and the disruptedalleles in the F1 heterozygotes. The heterozygous mice wereintercrossed, and their offspring were genotyped. Southern blot analysesshowed that the DNA hybridized with the 5′ and 3′ probes shown in FIG.1A and gave the signals expected from the wild-type (+/+), heterozygous(+/−), and homozygous-null (−/−) animals (FIG. 1B). After genotypingmore than 300 mouse tails, it was determined that 24% of the progenywere Acc2^(−/−), 22% were Acc2^(+/+), and 54% were Acc2^(+/−); theseresults are consistent with Mendelian inheritance. The Acc2^(−/−)mutants were viable, bred normally, and appeared to have a normal lifespan.

EXAMPLE 2

[0055] Acc2 Expression in Acc2^(−/−) Transgenic Mice

[0056] Northern blot analyses of total RNA of skeletal muscle tissuesresected from the wild-type, heterozygous, and homozygous-null animalsshowed no detectable Acc2 mRNA in the homozygous-null animals and, asexpected, the level of Acc2 mRNA in the heterozygous animals was half ofthat in the wild-type (FIG. 1C). Western blot analyses of heart,skeletal muscle, and liver tissues from the Acc2^(−/−) mutant mice usingavidin peroxidase to detect biotin-containing proteins showed noexpression of ACC2 protein (FIG. 1D). The levels of ACC2 protein (280kDa) were higher than those of ACC1 protein (265 kDa) in the heart andskeletal muscle tissues of the wild-type mice, whereas the ACC1 proteinwas more predominant in their liver tissues.

[0057] The absence of ACC2 protein in the Acc2^(−/−) mutant mice wasfurther confirmed by confocal immunofluorescence microscopic analysisusing affinity-purified anti-ACC2-specific antibodies (Abu-Elheiga, L.,W. R. Brinkley, L. Zhong, S. S. Chirala, G. Woldegiorgis, and S. Wakil.Proc Natl Acad Sci USA., 97:1444-1449, 2000). Whereas the hearts,skeletal muscles, and livers of the wild-type mice had abundantexpression of ACC2 antigen, there was no expression of this protein inthe Acc2^(−/−) mutant mice (data not shown). Thus, by all measurements,the Acc2^(−/−) mutant allele is a null allele.

EXAMPLE 3

[0058] Malonyl-CoA levels in Acc2^(−/−) Transgenic Mice

[0059] Since the levels of malonyl-CoA in animal tissues are attributedto the activities of both ACC1 and ACC2, the consequences of the absenceof ACC2 on the malonyl-CoA levels in these tissues and whether ACC1 cancompensate and, consequently, raise the levels of malonyl-CoA in thesetissues was determined. In comparing the liver tissues of the wild-typeand Acc2^(−/−) mutant mice, there were no significant differences in themalonyl-CoA levels and overall ACC activities, suggesting that almostall of the malonyl-CoA CoA in the liver is contributed by ACC1 (FIG. 2).

[0060] On the other hand, in comparing the skeletal muscle and hearttissues of the same two groups of mice, the levels of malonyl-CoA CoAwas found to be about 30- and 10-fold lower, respectively, in thesetissues of the Acc2^(−/−) mutant mice than in those of the wild-typemice. This suggests that ACC2 is the main contributor of malonyl-CoA inskeletal muscle and heart (FIG. 2).

[0061] During fasting, the levels of malonyl-CoA dropped comparably inthe liver tissues of both the wild-type and the Acc2^(−/−) mutant mice,suggesting that ACC1 is affected by dietary conditions (FIG. 2). Thelevels of malonyl-CoA in the heart and muscle tissues of the fastedAcc2^(−/−) mutant mice were very low, suggesting that ACC1 in thesetissues is also affected by diet (FIG. 2). Since malonyl-CoA in themuscle is generated primarily by ACC2 (Thampy, K. G., J Biol Chem.,264:17631-17634, 1989), starving the wild-type mice reduced its levelsby 70% from that in the muscles of the well-fed mice, suggesting thatthe ACC2 activity in these mice might be regulated by diet. ACC2activity may be significantly reduced by a decrease in the amount ofACC2 expressed or by down-regulation of its activity or by both.

EXAMPLE 4

[0062] Fatty Acid Accumulation in Acc2^(−/−) Transgenic Mice

[0063] Because the ACC reaction is the rate-determining step in fattyacid synthesis (Wakil, S. J., Stoops, J. K., and Joshi, V. C., Ann RevBiochem., 52:537-579, 1983) and the levels of malonyl-CoA in the liversof the wild-type and Acc2^(−/−) livers were similar, fatty acidsynthesis was also expected to be similar. Indeed, the synthesis ofpalmitate measured by the incorporation of [¹⁴C]-acetyl-CoA was the samefor both groups. However, the livers of wild-type mice were lighter incolor than the mutant livers, suggesting that they contained more fat(FIG. 3A).

[0064] To confirm this supposition, liver tissues were stained with OilRed-O to detect lipids and estimate their lipid and triglyceridecontents. Wild-type livers contained abundant lipid droplets (FIG. 3B),which are primarily triglycerides, whereas Acc2^(−/−) livers containedsignificantly fewer lipid droplets (FIG. 3C). Extraction and analysis ofthe total lipids by thin-layer chromatography showed that the mutantlivers contained 20% less lipid than wild-type livers, and thetriglyceride content of the lipid was 80% to 90% lower than wild-type.

EXAMPLE 5

[0065] ACC1 and ACC2 Modulate Distinct Pools of Malonyl CoA.

[0066] Since the activities of ACC and fatty acid synthase (FAS)activities in liver extracts of wild-type and Acc2^(−/−) mutants werethe same, the difference in the liver lipid content must be secondary touncontrolled mitochondrial fatty acid oxidation in the Acc2^(−/−) liversrather than due to a suppression of fatty acid synthesis. Also, becausemalonyl CoA is a negative regulator of the mitochondrial carnitinepalmitoyl-CoA shuttle system (McGarry, J. D., and N. F. Brown., Eur. J.Biochem., 244:1-14, 1997), its absence in Acc2^(−/−) livers would beexpected to increase fatty acid translocation across the mitochondrialmembrane and subsequent β-oxidation. Thus, these results suggest thatmalonyl-CoA, synthesized by ACC2, affects the accumulation of fat in theliver by controlling fatty acid oxidation. Since ACC1 -generatedmalonyl-CoA, which is abundant in the livers of both groups of mice,apparently did not inhibit the β-oxidation of fatty acids, it can beconcluded that the malonyl-CoA produced by ACC1 and ACC2 exists in twodistinct compartments of the cell—the cytosol and the mitochondria,respectively, and carries out distinct functions in these compartments.Because both ACC1 and ACC2 are present in both the periportal (zone 1)and perivenous (zone 3) hepatocytes of rat liver, it is unlikely thatthe two pools of malonyl-CoA were derived from differential expressionof ACC1 and ACC2 in these discrete regions of the liver.

EXAMPLE 6

[0067] Analysis of Glycogen in the Liver of Acc2⁻ transgenic mice

[0068] Glycogen, the storage form of glucose in the liver and muscles isan important regulator of energy homeostasis in animals includinghumans. Its synthesis and degradation is closely related to glucosemetabolism. The enzymes involved in glycogen metabolism are highlyregulated by hormones such as insulin, glucagon, and epinephrine.

[0069] To examine whether the loss of ACC2 affects the level ofglycogen, frozen sections of livers resected from wild-type andAcc2^(−/−) mutant mice were stained for glycogen (FIGS. 3D and 3E). Inthe nourished state, the wild-type livers contained abundant amounts ofglycogen (410±10 μmol/g of wet tissue), whereas the livers of Acc2^(−/−)mice contained 20% less glycogen (325±14 μmol/g of wet tissue).Speculation suggests that more glucose is utilized in the synthesis offatty acids and their subsequent oxidation in the Acc2^(−/−) liver, thusdepleting glycogen. In the 24-hour-fasted wild-type mouse liver,glycogen is clearly present (FIG. 3D), whereas it was undetectable inthe Acc2^(−/−) mutant liver (FIG. 3E).

EXAMPLE 7

[0070] Analysis of Blood Glucose and Lipids in Acc2^(−/−) TransgenicMice

[0071] The next step was analysis of the serum levels of cholesterol,glucose, triglycerides, free fatty acids and ketone bodies in wild-typeand Acc2^(−/−) mice fed a standard diet. Cholesterol levels were similarin both groups of mice (92.8±3.1 and 95.1±7.4 mg/dl), and glucose levelswere 20% lower in mutant mice (176.6±6.5 versus 136.2±5.4 mg/dl). Fattyacid levels were lower in mutant mice (1.37±0.31 versus 0.84±0.12 mM),whereas triglyceride levels were 30% higher in the mutant mice (35.1±2.5versus 45.2±5.9 mg/dl), possibly due to mobilization of triglyceridesand fatty acids from liver and/or adipose for their delivery to theheart and muscles as a substrate for oxidation. Serum levels of theketone bodies (P-hydroxybutyrate) were nearly undetectable in both thewild-type and mutant mice. However, an overnight fast (10 to 12 hours)increased the blood β-hydroxybutyrate concentration of the Acc2^(−/−)mice fourfold over that of the wild type (2.5±0.6 mM versus 0.7±0.0.5mM, n=5), consistent with a higher degree of fatty acid oxidation in themutant mice.

EXAMPLE 8

[0072] Fatty Acid Oxidation in Acc2^(−/−) Transgenic Mice

[0073] To provide further evidence for the role of ACC2-synthesizedmalonyl-CoA as the regulator of fatty acid oxidation, fatty acidoxidation was investigated in the mouse soleus muscle, a type II muscletissue responsive to hormonal regulation (Vavvas, D., Apazidis, A.,Saha, A. K., Gamble, J., Patel, A., Kemp, B. E., Witters, L. A., andRuderman, W. B., J Biol Chem., 272:13255-13261 1997; Alam, N., and E. D.Saggerson. Biochem J., 334:233-41, 1998; Abu-Elheiga, L., Jayakumar, A.,Baldini, A., Chirala, S. S., and Wakil, S. J. Proc Natl Acad Sci. USA92, 4011-4015, 1995; Abu-Elheiga, L., Almarza-Ortega, D. B., Baldini,A., and Wakil, S. J. J Biol Chem. 272, 10669-10677, 1997; —Ha, J., J. K.Lee, K.-S. Kim, L. A. Witters, and K.-H. Him. Proc Natl Acad Sci USA.93:11466-11470, 1996; Rasmussen, B. B. and Wolfe, R. R., Ann. Rev. Natr.19:463, 1999; and, Bressler, R. and Wakil, S. J. J Biol Chem.236:1643-1651, 1961).

[0074] As shown in FIG. 4, the oxidation of [³H]palmitate was 30% higherin the isolated soleus muscles of Acc2^(−/−) mutant mice than in thoseof the Acc2^(+/+) mice. Insulin is known to activate both ACC1 and ACC2and, thereby, to induce fatty acid synthesis and inhibit fatty acidoxidation, respectively. Adding insulin to soleus muscles resected fromwild-type and from Acc2^(−/−) mutant mice did not affect fatty acidoxidation in the Acc2^(−/−) mutant muscle cells (FIG. 4) but did reducepalmitate oxidation by about 45% in the wild-type muscle cells (FIG. 4).Based on these results, it can be concluded that the insulin-mediatedinhibition of β-oxidation occurs through the activation of ACC2,probably by dephosphorylation (Lopaschuk, G., and Gamble, J. Can JPhysiol Pharmacol. 72:1101-1109. 1994; Kudo, N., Bar, A. J., R. L.,Desai, S., Lopaschuk, GD. J Biol Chem. 270:17513-17520, 1995; Dyck, J.R., N. Kudo, A. J. Barr, S. P. Davies, D. G. Hardie, and G. D.Lopaschuk. Eur J Biochem. 262:184-190, 1999; Vavvas, D., Apazidis, A.,Saha, A. K., Gamble, J., Patel, A., Kemp, B. E., Witters, L. A., andRuderman, W. B. J Biol Chem. 272:13255-13261, 1997; Iverson, A. J., A.Bianchi, A. C. Nordlund, and L. A. Witters. Biochem J. 269:365-371,1990; Kim, K. H., F. Lopez-Casillas, D. H. Bai, X. Luo, and M. E. Pape.Faseb J. 3:2250-2256, 1989; Thampy, K. G., and Wakil, S. J. J. Biol.Chem. 263, 6454-6458, 1988; Mabrouk, G. M., Helmy, I. M., Thampy, K. G.,and Wakil, S. J. J. Biol. Chem. 265, 6330-6338, 1990; Mohamed, A. H., W.Y. Huang, W. Huang, K. V. Venkatachalam, and S. J. Wakil. J Biol Chem.269:6859-6865. 1994; and, Hardie, D. G. Prog Lipid Res. 28:117-146,1989).

[0075] The role of ACC2 in the regulation of mitochondrial oxidation offatty acids was further confirmed by using isoproterenol, an analog ofglucagon, which produces effects opposite of those of insulin. Addingisoproterenol to wild-type soleus muscle increased palmitate oxidationby 50% (FIG. 4), raising it to nearly the same level as that found inthe mutant muscle cells. It is noteworthy that isoproterenol alsofurther increased fatty acid oxidation in the mutant soleus muscle cells(FIG. 4). This additional increase may be due to factors independent ofmalonyl-CoA (Kim, K. H., F. Lopez-Casillas, D. H. Bai, X. Luo, and M. E.Pape. Faseb J. 3:2250-2256, 1989).

[0076] Altogether, these results confirm for the first time thatmitochondria-associated ACC2, and not cytosolic ACC1, is responsible forthe insulin-mediated activation and isoproterenol (glucagon)-mediatedinactivation that results, respectively, in decreased and increasedfatty acid oxidation. Since the mitochondrial CPTI activities of thesoleus muscles from both groups of mice were very similar (data notshown), the observed effects of these hormones are solely due to theireffect on ACC2.

EXAMPLE 9

[0077] Feeding Experiments with Acc2^(−/−) Transgenic Mice

[0078] It appears that the mitochondrial β-oxidation of fatty acidsoccurs in the Acc2^(−/−) mutant mice in an unregulated yet sustainedmanner. To investigate the role of this type of fatty acid β-oxidationand its effect on food consumption and weight gain, feeding experimentswere performed with three groups of mice (each group consisting of 5wild-type and 5 Acc2^(−/−) mutant mice) that were fed a weighed standarddiet ad liberatum. (FIG. 5 represents a plot of one of the groups). Foodconsumption (no spillage was noted) for each group was measured everyweek for 27 weeks, and the weight of each mouse was recorded weekly.

[0079] On the average, each Acc2^(−/−) mutant mouse consumed 20-30% morefood per week than the wild-type mice (FIG. 5A) and maintained anaverage body weight of 21 g compared to 23 g per wild-type mouse. TheAcc2^(−/−) mutant mice were generally leaner, weighing about 10% lessthan the wild-type mice throughout the feeding periods (FIG. 5B). Inaddition, Acc2^(−/−) mutant mice accumulated less fat in their adiposetissues (FIGS. 5C and 5D). For example, the epididymal fat pad tissue inan Acc2^(−/−) male weighed 0.75 g as compared to 1.4 g in a wild-typemale littermate (FIG. 5E). The decrease in the adipose size resulted ina decrease in the leptin released to the plasma from 53±9 ng/ml in thewild-type mice to 36±3 ng/ml in the mutant mice. Thus, mitochondrialβ-oxidation of fatty acids regulates fat storage in the adipose tissue.

EXAMPLE 10

[0080] Generation of Acc1^(−/−) Transgenic Mice

[0081] To demonstrate the importance of ACC1 in the de novo synthesis offatty acids, the same strategy was followed to generate an Acc1-knockoutmouse as done for Acc2. Like ACC2, the ACC1 isoform is also highlyconserved among animal species (Thampy, K. G. J Biol Chem.264:17631-17634, 1989). A forward primer (5′-GGATATCGCATCACAATTGGC-3′)(SEQ ID No. 3) based on the human Acc1 CDNA and a reverse primer(CCTCGGAGTGCCGTGCTCTGGATC-3′) (SEQ ID No. 4) that contained thebiotin-binding site was designed and used to amplify a 335-bp cDNA probeusing human cDNA as a template. A 129/SvEv mouse genomic library wasscreened with the PCR fragment as described for ACC2, and a 14-kbp clonewas isolated, mapped with restriction enzymes, and analyzed by Southernblotting (FIG. 6B). A correctly targeted clone (FIG. 6A) wasmicroinjected into C57BL/6J mouse blastocysts, which were then implantedinto the uterine horns of pseudopregnant female mice. The male chimerasthus generated were bred with C57BL/6J mates, and the Acc1 heterozygousoffspring were interbred.

[0082] After analyzing genomic DNA from more than 300 progenies bySouthern blotting using both the 5′ and 3′ probes, homozygous Acc1 -nullmutant offspring were not obtained. The litter sizes were less thanaverage, being 6 or 7, and 35% of the progeny were wild-type and 65%were heterozygous. These results demonstrate that the Acc1 mutation isembryonically lethal.

[0083] To characterize this embryonic lethality, the mating of theheterozygotes was timed and the resulting embryos were genotyped. Atgestation days E12.5 and E13.5, the viable embryos were 35% wild-typeand 65% heterozygous, indicating that the lethality had occurredearlier. At gestation day E9.5, the remains of dead embryos wererecovered, and at gestation day E8.5, degenerating embryos wererecovered from inside the ectoplacental cone.

[0084] Discussion

[0085] Obesity is a major health factor that affects the body'ssusceptibility to a variety of diseases such as heart attack, stroke,and diabetes. Obesity is a measure of the fat deposited in the adiposein response to food intake, fatty acid and triglyceride synthesis, fattyacid oxidation, and energy consumption. Excess food provides not onlythe timely energy needs of the body, but promotes glycogen synthesis andstorage in liver and muscle and fatty acid and triglyceride synthesisand storage in the fat tissues. Calorie restriction or starvationpromotes glycogenolysis that supplies glucose where needed and lipolysisthat supplies fatty acids for oxidation and energy production. Insulinand glucagon are the hormones that coordinate these processes.Malonyl-CoA is the key intermediate in fatty acid synthesis and hasrecently assumed an additional role as a second messenger that regulatesenergy levels (ATP) through fatty acid oxidation, which in turn affectsfatty acid synthesis and carbohydrate metabolism.

[0086] The studies described above provide a definitive characterizationof the role of malonyl-CoA produced by ACC2 in the regulation of fattyacid oxidation and energy metabolism. Malonyl-CoA generated by ACC1 isthe donor of the C₂ units required for fatty acid synthesis. Acetyl CoA,the substrate for ACC1 and ACC2, is the product of pyruvate oxidation,hence studies of the carboxylases interrelate three major metabolicpathways—carbohydrate metabolism, fatty acid synthesis, and fatty acidoxidation.

[0087] Studies on animal carboxylases, usually a mixture of ACC1 andACC2, showed that these enzymes are under long-term control at thetranscriptional and translational levels and under short-term regulationby phosphorylation/dephosphorylation of targeted Ser residues and byallosteric modifications by citrate or palmitoyl-CoA. Several kinaseshave been found to phosphorylate both carboxylases and to reduce theiractivities. Insulin activates the carboxylases through theirdephosphorylation, whereas glucagon and epinephrine inactivate them as aresult of their phosphorylation. The AMP-activated protein kinase(AMPK), one of the most notable kinases, is activated by a high level ofAMP concurrent to a low level of ATP through mechanisms involvingallosteric regulation and phosphorylation by protein kinase (AMPKkinase) in a cascade that is activated by cellular stressors thatdeplete ATP. Through these mechanisms, when metabolic fuel is low andATP is needed, both the ACC activities are turned off byphosphorylation, resulting in the low malonyl-CoA levels that lead toincreased synthesis of ATP through increased fatty acid oxidation anddecreased consumption of ATP for fatty acid synthesis.

[0088] The differential expression of ACC1 and ACC2 in varioustissues—ACC1 is highly expressed in liver and adipose and ACC2 ispredominant in heart and muscle—and their cellular localization—ACC1 inthe cytosol and ACC2 on the mitochondrial membrane—suggest that theirfunctions are different though interrelated. The cytosolic ACC1-generated malonyl-CoA is utilized by the fatty acid synthase, whichalso is a cytosolic enzyme, for the synthesis of fatty acids. Themitochondrial ACC2-generated malonyl-CoA functions as a regulator ofCPTI activity—CPTI being the first enzyme that catalyzes the shuttlingof long-chain fatty acids into the mitochondria for β-oxidation andenergy production. ACC2-generated malonyl-CoA, therefore, is a secondmessenger that regulates ATP levels through fatty acid oxidation, which,in turn, affects fatty acid synthesis and carbohydrate metabolism.

[0089] The present studies of the Acc2 mutant mice strongly support thisconclusion. The levels of malonyl-CoA in the livers of the mutant micewere similar to those in the livers of the wild-type mice, indicatingits synthesis by ACC1, the predominant carboxylase in this tissue. Inthe livers of the wild-type mice, the malonyl-CoA is used to synthesizefatty acids, which are then converted into triglycerides that accumulateas lipid droplets (FIG. 3A). In the livers of the Acc2^(−/−) mutantmice, the uncontrolled CPTI activity results in the oxidation of fattyacids by the liver mitochondria or in the conversion of fatty acids intolipids (very-low-density lipoproteins), which are then transportedthrough the bloodstream to the heart and muscles to overcome theincreased demand of these tissues for fatty acids consequential touninhibited CPTI activity and amplified fatty acid oxidation. Theseconclusions were supported by the near absence of malonyl-CoA in theheart and skeletal muscle tissues of the Acc2^(−/−) mutant mice, by thehigher fatty acid-oxidation rate in the soleus muscles of the Acc2^(−/−)mutant mice, and by the occurrence of fatty acid oxidation independentof insulin and isoproterenol, an analog of glucagon (FIG. 4).

[0090] Finally, knocking out ACC2 in mice has demonstrated that the lackof malonyl-CoA, the mitochondrial second messenger, produces offspringthat exhibit increased oxidation of fatty acids, decreased accumulationof lipids, and decreased storage of glycogen in the liver but are stillmorphologically normal, grow at an expected rate, and breed normally(their longevity and aging are being followed). All of the metabolicchanges are expressed in food consumption patterns and body weight—theAcc2^(−/−) mutant mice who were fed a standard diet typically consumed20% more food than did the wild-type mice yet eventually lost 10% oftheir body weight.

[0091] The reduction in fat content and the size of the adipose tissueled to a reduction of about 30% in leptin released to the plasma,similar to that occurring in fasted mice. This signaled the hypothalamusto produce the appetite-stimulating neuropeptide Y, which promotesfeeding. This is the most plausible explanation for the observation thatAcc2^(−/−) mice have smaller fat stores even as they consumed more foodthan the wild-type mice (FIGS. 5A-5E). It has been suggested thatmalonyl-CoA may play a role in signaling the availability ofphysiological fuel by acting through the hypothalmic neurons. Thissuggestion was based on the inhibition of ACC by 5-(tetradeculoxy)-2furoic acid that increases food uptake in mice treated with fatty acidsynthase inhibitors. Although this possibility could not be ruled out inthe Acc2^(−/−) mice, the lower leptin levels in the plasma may besufficient to increase appetite. Moreover, the Acc2^(−/−) mice appear tobe normal, with no obvious neurological abnormalities.

[0092] Maintenance of high levels of fatty acid oxidation results inreduced fat accumulation and storage, a physiological state that humanstry to attain through exercise. Pharmacological inhibition of ACC2 mayallow individuals to lose weight while maintaining normal caloricintake.

1 4 1 25 DNA Artificial sequence Reverse oligonucleotide primer for thePCR amplification of the biotin-binding region of ACC2 1 ctgaatgatggggggctcct gctct 25 2 25 DNA Artificial sequence Reverse oligonucleotideprimer for the PCR amplification of the biotin-binding region of ACC2 2ttcagccggg tggactttag caagg 25 3 21 DNA Artificial sequence Forwardoligonucleotide primer for the PCR amplification of the biotin-bindingregion of ACC1 3 ggatatcgca tcacaattgg c 21 4 24 DNA Artificial sequenceReverse oligonucleotide primer for the PCR amplification of thebiotin-binding region of ACC1 4 cctcggagtg ccgtgctctg gatc 24

What is claimed is:
 1. A method of promoting fatty acid oxidation and weight loss in an individual, comprising the step inhibiting the activity of acetyl-CoA carboxylase 2 in said individual.
 2. The method of claim 1, wherein said activity is inhibited by administration of an inhibitor of acetyl-CoA carboxylase 2 (ACC2) to said individual.
 3. The method of claim 1, wherein said individual has a pathophysiological condition.
 4. The method of claim 3, wherein said pathophysiological condition is selected from the group consisting of obesity and diabetes.
 5. A method of decreasing blood sugar in an individual, comprising the step of administering an inhibitor of acetyl-CoA carboxylase 2 (ACC2) to said individual.
 6. The method of claim 5, wherein said individual has diabetes.
 7. A transgenic mouse, said mouse comprising a mutation in an endogenous ACC2 gene for the acetyl-CoA carboxylase 2 isoform of acetyl-CoA carboxylase, wherein said mutation inactivates said gene and results in the lack of expression of a functional acetyl-CoA carboxylase 2 isoform.
 8. The mouse of claim 7 wherein one or more exons of said ACC2 gene has been deleted.
 9. The mouse of claim 8, wherein said exons have been replaced with heterologous DNA sequences.
 10. The mouse of claim 9, wherein said heterologous DNA sequences comprise an hypoxanthine phosphorylribosyltransferase expression cassette.
 11. The mouse of claim 10, wherein an exon encoding a biotin binding motif of ACC2 is replaced with an hypoxanthine phosphorylribosyltransferase expression cassette.
 12. The mouse of claim 7, wherein said mouse exhibits a phenotype comprising a metabolic reduction in malonyl-CoA production in skeletal muscle and heart.
 13. The mouse of claim 12, further comprising a phenotype of unrestricted fat oxidation and reduced fat accumulation in the liver and fat storage cells.
 14. The mouse of claim 13, further comprising a phenotype of consuming more calories than a wild-type mouse, yet accumulating less fat than a wild-type mouse.
 15. A method of screening for an inhibitor of acetyl-CoA carboxylase 2 isoform activity comprising the steps of: administering potential inhibitors to wild-type mice; and, screening for mice which exhibit the phenotype of the transgenic mouse of claim
 14. 16. An acetyl-CoA carboxylase 2 inhibitor identified by the method of claim
 15. 17. A pharmaceutical composition comprising the acetyl-CoA carboxylase 2 inhibitor of claim 16 and a pharmaceutically acceptable carrier.
 18. A method of obtaining a purified preparation of acetyl-CoA carboxylase 1 protein which is free of acetyl-CoA carboxylase 2 comprising the step of: purifying said acetyl-CoA carboxylase 1 protein from tissues obtained from the transgenic mouse of claim
 7. 19. A method of obtaining murine antibodies against acetyl-CoA carboxylase 2 which are less crossreactive with acetyl-CoA carboxylase 1 and other mouse proteins comprising the step of: generating said antibodies in the transgenic mouse of claim
 7. 20. A cell line derived from the transgenic mouse of claim
 7. 21. The cell line of claim 20, wherein said cell line is derived from cells selected from the group consisting of muscle cells, heart cells, adipose cells, and liver cells.
 22. A method of screening for agonists and antagonists of ACC2 comprising the steps of: administering a candidate compound to the cell line of claim 20 and to cell lines derived from wild-type mice; and, monitoring said cell lines for alterations in cellular activity, wherein a compound that specifically acts on ACC2 will have altering cellular activity in wild-type cells but will have no effect on the cell line of claim
 20. 23. The method of claim 22, wherein monitored cellular activities are selected from the group consisting of mRNA expression, protein expression, protein secretion, and lipid metabolism. 